The IVEM automation system consists of a cooled, slow-scan, 1024x1024-pixel CCD camera, an automated goniometer, an OS-9/VME bus computer system, and specialized software for automated tomography. The system was purchased from the BMIRR grant from Tietz Video and Image Processing Systems, Gauting Germany, and installed in FY 1996. For each image in a tilt series, the system automatically tilts the specimen by a preset increment, re-focuses the image, re-centers the object of interest, and records the image using the CCD or film. Both automatic focusing and automatic re-centering of the object of interest are done using cross-correlation and peak search algorithms, and are done adjacent to the position on the specimen at which the tilt images are being recorded. This makes possible minimum-dose tomography, essential for irradiation-sensitive cryo specimens. The major task in FY 1997 was to modify the automation software to work optimally with our microscope. The reliability of tomographic recording was improved by implementing automatic compensation for the change of illumination size and position caused by focus change, and also by automatically correcting the shift of the reference image to compensate for specimen drift that occurs while cross-correlation and peak search operations are taking place. Both of these compensations make the cycle time for recording each tilt image a bit longer. The focus change compensation is needed because there is excessive focus change during tilting. If the goniometer problems described under "IVEM operation" can be solved, compensation may not be needed. At present, the compensations are not always sufficient avoid the need for occasional manual corrections during automated topography. If the goniometer problem cannot be solved, we have determined how to make the needed additional corrections, which involve corrections of focus-induced rotation of the beam tilt and shift, and image shift axes. The speed of automated tomography has been increased by reducing the number of communications between the Tietz computer and the microscope's internal computer. This was possible because the software was originally written for Philips microscopes, which require a read of the present state before changing any microscope parameter, while JEOL microscopes accept absolute setting of any parameter. The increased speed means that, in some cases (i.e. without the compensations described above), the cycle time for each tilt image is 2 min instead of 4 min. Low-magnification capability was added to the tomography software. This was necessary because the magnification on the CCD camera is twice the nominal microscope magnification, so some structures are too large to record as a single CCD image. A lower-magnification image is also useful when there is a large shift of the specimen position after tilting, which needs to be automatically corrected. The normal "low magnification" mode is not useable for automated tomography because of objective lens hysteresis effects (thermal and magnetic) in switching between low and normal magnification ranges. Therefore a calibrated lens-control program for the three lowest projector lenses was developed to decrease the magnification for CCD imaging by up to 1/5th. The automation software was also modified to make it easier to take individual low-dose images, to make possible film recording during automated tomography (instead of or in addition to CCD recording), and to do rapid semi-automated tomography with film or CCD recording. Using semi-automation, we can take a full double-tilt series of 122 images in only 3 hours, although absolute minimum-dose recording is not achieved. Six to eight hours is needed for a double-tilt series if full automation is used, and all compensations are applied. We can also do semi-automated tomography using a program we have written for a VAX workstation. This serves as a backup for the Tietz system. The VAX workstation is also used to monitor data transferred from the Tietz system to the SGI computers which used for reconstruction, and to show displays of results at the frequent tours of the IVEM. We will need to replace this workstation with a well-equipped PC since neither the workstation nor the VMS operating system required will maintained in 1998. The images recorded on the CCD are not of sufficient resolution for many projects, which require high-resolution reconstruction of large objects. The actual resolution of the 1024x1024 CCD image is only about 700x700 pixels at best, due to the compromise we had to make in choosing the phosphor thickness and grain size of our scintillator to give the sensitivity needed for minimum-dose work. Thus, at present we use film recording for some projects. This is inefficient and expensive. We believe that we can obtain the needed resolution in the digital images in two ways: recording the image in the STEM mode (see TRD "IVEM operation and development"), and/or automatically montaging four CCD images. In both cases, a final image of 2048x2048 pixels can be obtained. In both cases, the alternative imaging method will have to be integrated into the tomography system. It will be necessary to purchase an A/D converter board for the Tietz system to acquire 12-bit STEM images, an unanticipate d expense of $3500. We have successfully record tomographic tilt series on frozen-hydrated, plunge-frozen isolated triad-junction vesicles embedded in a 0.1lm layer of vitreous ice (see TRD "Structure of the triad junction"). The original pixel size was 0.7nm, and the 3-D resolution was about 3-4nm. We hope to try 0.25lm thick cryo-sections soon. Double-tilt reconstructions are not possible with cryo specimens due to unavailability of a tilt-rotation cryo-transfer stage, thus the time required to acquire a minimum-dose series is short enough, even with full compensations, to avoid having to refill the liquid nitrogen Dewar of the cryo-transfer holder. Rath, B.K., M. Marko, M. Radermacher and J. Frank. 1997 Low-dose automated electron tomography: A recent implementation. J. Structural. Biology (in press) Marko, M., Buttle, K.F., Frank, J., Khodjakov, A., Mannella, C.A., McEwen, B.F., Rath, B.K., Rieder, C.L. (1997) Electron Tomography of Cellular Organelles, a Sampling of Recent Results Cell Vision 4:(2), 137-138.